Role of cell surface HSPGs in monocyte adhesion to Cyr61.

(A) Isolated monocytes (open bars) or 1064SK fibroblasts (closed bars) were preincubated with the indicated concentrations of heparin for 30 minutes at 37°C and added to wells coated with 10 μg/mL Cyr61. Cell adhesion proceeded for 20 minutes at 37°C. Adherent monocytes were quantified by measuring A₄₅₀ in the acid phosphatase assay. Adherent fibroblasts were fixed, stained with 1% methylene blue, and the extracted dye was quantified by measuring A₆₅₀. (B) Cells were pretreated with or without 2 U/mL heparinase I for 30 minutes at 37°C and allowed to adhere to Cyr61-coated wells for 20 minutes at 37°C. (C) Amino acid sequence of wild-type Cyr61 (Cyr61-WT) at residues 278-314 with the 2 heparin-binding motifs underlined. The Cyr61 mutant protein (Cyr61-DM) contains mutations in both motifs where basic residues were substituted with alanine as shown by the bold letters. ADP-activated monocytes were allowed to adhere to microtiter wells coated with the indicated concentrations of Cyr61-WT or Cyr61-DM. Data shown are means of triplicate determinations and error bars represent SEs. Figures are representative of 3 experiments.